An established hallmark of tumorigenesis is the biosynthesis of aberrant glycan chains due to changes in the expression of glycoprocessing enzymes in tumor tissue. These aberrations become more marked as the tumor acquires a more aggressive phenotype. Tumor cell-surface carbohydrates play important roles in the motility and metastasis of many different cancer cells. In addition, many of these aberrant glycans are tumor-associated carbohydrate antigens (TACA) and have been used in the development of tumor vaccines. Since most of the cellular interactions with TACAs are not well understood, there is an urgent need to better characterize the specific molecular interactions that occur during these events. One feature of carbohydrate binding to macromolecules that is well understood is the concept of multivalency: Monomer carbohydrates bind to proteins very weakly while clustering of a monomer raises this affinity as much as a million-fold. We have prepared the important Thomsen-Friedenreich (Tf) antigen (Gal(beta)1-3GalNAc(alpha)-O-Ser/Thr) on very specific templates to take advantage of this so-called cluster glycoside effect. As mentioned in the last report, we have prepared gold self-assembled nanospheres and quantum dots containing sugar derivative and reported preliminary details on their function. The in vivo experiments with our gold nanospheres in mice were repeated twice with varying results. However, these were caused by the use of a tumor cells that had had a different genetic makeup than the original and an error in the amount of tumor used. We have done further in vitro characterization of the gold particles and found them to act differently than monovalent sugar molecules. A large in vivo study has yielded conflicting but highly provocative results with varying concentrations of particles being tested against control (linker-only) nanoparticles. We seem to be able to inhibit primary tumor growth in vivo, but we possibly promote metastasis with particles of larger hydrodynamic volume. In the past year, a concentrated research effort was mounted to understand the conflicting in vivo data. We feel we have solved this problem by closely examining the size distribution, surface density of sugars and the linker technology of the nanoparticles. We have shown that a specific stereochemistry of the saccharide is detrimental to particle self assembly and solved this problem with reduced surface density of the carbohydrate. We have also shown that specific surface densities of simple sugars can either promote or inhibit the attachment of HIV to certain mammalian cells. We are optimizing this technology to find the most robust particles for in vivo applications. In the last report we reported synthesis of mucin glycopeptides and developed new linker technology to attach these to gold particles. We discovered this year that that linker technology was not synthetically feasible due to unanticipated aggregation effects. We have prepared a much more robust linker and prepared 4 glycopeptides plus a control peptide that were linked to gold nanoparticles. Characterizations were performed and the particles were uniform. We have started a vaccination protocol with our collaborator Kate Rittenhouse Olsen on eight mice with two separate particles containing MUC4 repeating unit glycopeptides. In addition, we are exploring the nature of the TF antigen by preparing particles with amino acid-linker technology to compare with those of simple saccharides and linker alone. We are very excited about the fate of the vaccination studies since this would be a novel platform to present antigen to a host.